As X. Vasicola Pv. Arecae Comb

ORE Open Research Exeter

TITLE Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.

AUTHORS Studholme, DJ; Wicker, E; Abrare, SM; et al.

JOURNAL Phytopathology

DEPOSITED IN ORE 24 January 2020

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http://hdl.handle.net/10871/40555

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A NOTE ON VERSIONS

The version presented here may diﬀer from the published version. If citing, you are advised to consult the published version for pagination, volume/issue and date of publication Phytopathology • XXXX • XXX:X-X • https://doi.org/10.1094/PHYTO-03-19-0098-LE

Letters to the Editor

Transfer of Xanthomonas campestris pv. arecae and X. campestris pv. musacearum to X. vasicola (Vauterin) as X. vasicola pv. arecae comb. nov. and X. vasicola pv. musacearum comb. nov. and Description of X. vasicola pv. vasculorum pv. nov.

David J. Studholme,1,† Emmanuel Wicker,2 Sadik Muzemil Abrare,3 Andrew Aspin,4 Adam Bogdanove,5 Kirk Broders,6 Zoe Dubrow,5 Murray Grant,7 Jeffrey B. Jones,8 Georgina Karamura,9 Jillian Lang,10 Jan Leach,10 George Mahuku,11 Gloria Valentine Nakato,12 Teresa Coutinho,13 Julian Smith,4 and Carolee T. Bull14 1 Biosciences, University of Exeter, Exeter, U.K. 2 IPME, University of Montpellier, CIRAD, IRD, Montpellier, France 3 Southern Agricultural Research Institute (SARI), Areka Agricultural Research Center, Areka, Ethiopia 4 Fera Science Ltd., York, U.K. 5 Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, 334 Plant Science Building, Ithaca, NY 14853, U.S.A. 6 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, U.S.A. 7 School of Life Sciences, Gibbet Hill, University of Warwick, Coventry, CV4 7AL, U.K. 8 University of Florida, Plant Pathology Department, 1453 Fifield Hall, Gainesville, FL 32611, U.S.A. 9 Bioversity International, Uganda 10 Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, U.S.A. 11 International Institute of Tropical Agiculture (IITA), East Africa Hub, IITA-Tanzania, P.O. Box 34441, Dar es Salaam, Tanzania 12 International Institute of Tropical Agriculture (IITA), Plot 15B, Naguru East Road, Upper Naguru, P.O. Box 7878, Kampala, Uganda 13 Department of Microbiology and Plant Pathology, Centre for Microbial Ecology and Genomics (CMEG), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Private Bag X28, Pretoria 0028, South Africa 14 Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA, U.S.A. Accepted for publication 5 July 2019.

ABSTRACT

We present an amended description of the bacterial species Xantho- share more than 98% average nucleotide with each other and with the type monas vasicola to include the causative agent of banana Xanthomonas strain. Our analyses and proposals should help to resolve the taxonomic wilt, as well as strains that cause disease on Areca palm, Tripsacum grass, confusion that surrounds some of these pathogens and help to prevent sugarcane, and maize. Genome-sequence data reveal that these strains all future use of invalid names.

The aim of this letter is to resolve the taxonomy of several There is confusion surrounding the taxonomy of some of these bacterial lineages that phylogenetically fall within the species lineages, not least around the previous splitting of Xanthomonas Xanthomonas vasicola Vauterin et al. 1995. These lineages include campestris pv. vasculorum into two new taxa: X. axonopodis pv. an economically important pathogen of banana and enset (X. vasculorum and [X. vasicola pv. vasculorum] (Vauterin et al. 1995). campestris pv. musacearum Yirgou and Bradbury 1968), a pathogen The latter name currently has no standing in the nomenclature, yet of Areca palm (X. campestris pv. arecae Rao and Mohan 1970) and has nevertheless been widely adopted. Our first objective is to closely related bacteria isolated from Tripsacum grass. Also within formally transfer these lineages into the species X. vasicola.A scope is a subset of X. campestris pv. vasculorum (Cobb 1894) Dye second objective is to formally propose X. vasicola pv. vasculorum 1978 from sugarcane and maize. Finally, it includes strains from according to the International Standards for Naming Pathovars of maize assigned to a taxon with an invalid name, [X. campestris pv. Phytopathogenic Bacteria (Young et al. 2001), which hereafter we zeae]. In this manuscript, pathovar names that have no standing in call the Standards. Our description of this pathovar corresponds to nomenclature are presented with square brackets ([]) as is standard the previous proposal (Vauterin et al. 1995) but also incorporates (Bull et al. 2012). [X. campestris pv. zeae] and agrees closely with the adopted usage of this name by the community. Despite a previous proposal (Vauterin et al. 1995), the name [X. vasicola pv. vasculorum] was † Corresponding author: D. J. Studholme; [email protected] excluded from the Names of Plant Pathogenic Bacteria (Younget al. 1996) due to the lack of a pathotype with an adherent description for Funding: This work was supported by the European Cooperation in Science and Technology (COST) Action CA16107 EuroXanth. the pathovar. Therefore, the name [X. vasicola pv. vasculorum] currently has no standing in the nomenclature and needs to be *The e-Xtra logo stands for “electronic extra” and indicates that one supplementary proposed as a pv. nov. Only a subset of X. campestris pv. vasculorum figure is published online. strains fall within X. vasicola (while others fall within X. axonopodis, The author(s) declare no conflict of interest. including the pathotype strain). Therefore, the problem cannot be solved by transferring X. campestris pv. vasculorum into X. vasicola Copyright © 2019 The Author(s). This is an open access article as a comb. nov. because no name-bearing strain is being transferred distributed under the CC BY 4.0 International license. into X. vasicola.

1 Members of the genus Xanthomonas, within the gamma- and colleagues distinguish type-A strains from type-B by sodium Proteobacteria, collectively cause disease on more than 400 plant dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) species (Hayward 1993), though some members are apparently of proteins, gas chromatography of fatty acid methyl esters and nonpathogenic (Garita-Cambronero et al. 2016; Vauterin et al. DNA-DNA hybridization (Yang et al. 1993). Type-A and type-B 1996; Vicente et al. 2017) and some have been isolated from clinical strains can also be distinguished by PCR-restriction fragment samples such as skin microbiota (Seite´ et al. 2017). Historically, length polymorphism analysis (Destefano´ et al. 2003). Table 1 lists taxonomy of Xanthomonas was tied to the host of isolation (Starr examples of X. campestris pv. vasculorum (Cobb 1894) Dye 1978 1981; Wernham 1948), with the genus being split into large strains that were classified in one or more of those studies. Vauterin numbers of species, each defined by this single phenotypic feature and colleagues assigned type-A strains to X. axonopodis pv. (Dye 1962). Subsequently, most of the species were transferred vasculorum (Cobb) Vauterin, Hoste, Kersters & Swings and (i.e., “lumped”) into a single species, X. campestris, and designated assigned type-B (Vauterin et al. 1995) to [X. vasicola pv. as nomenspecies because the organisms could not be distinguished vasculorum]. The type strain of X. campestris pv. vasculorum from one another by phenotypic and physiological tests (Dye and (Cobb 1894) Dye 1978 was among those assigned to X. axonopodis Lelliott 1974; Lapage et al. 1992). As a temporary solution, and to pv. vasculorum (Cobb) Vauterin, Hoste, Kersters & Swings. help to maintain a connection with the historical and plant- The names X. vasicola and X. vasicola pv. holcicola are listed in pathological literature, these nomenspecies were designated as the Comprehensive List of Names of Plant Pathogenic Bacteria pathovars within X. campestris, each defined by host range or (Bull et al. 2010; Young et al. 1996). However, as pointed out in disease syndrome (Dye et al. 1980). More recently, DNA sequence Bergey’s Manual (Brenner and Staley 2005), the name [X. vasicola comparisons and biochemical approaches revealed that in pv. vasculorum] (Vauterin et al. 1995) was excluded from the _ X. campestris, pathovar designation does not always correlate with Names of Plant Pathogenic Bacteria 1864 1995 (Young et al. 1996) phylogeny (Parkinson et al. 2007, 2009; Rodriguez-R et al. 2012). on the grounds that it was defective in terms of the Standards (Young There have been heroic advances to improve the taxonomy of the et al. 2001). Specifically, it lacked a modern description genus as a whole (Rademaker et al. 2005; Vauterinet al. 1990, 1995, differentiating it from other pathovars (Young et al. 2004) and 2000) and of individual taxa (Constantin et al. 2016; da Gama et al. lacked designation of a pathotype strain (Young et al. 1996). This is 2018; Jones et al. 2004; Timilsina et al. 2019; Trebaol´ et al. 2000), understandable given that Vauterin and colleagues were primarily based on phenotypic, chemotaxonomic, and genotypic analyses. concerned with species-level reclassification of the genus rather But in a number of taxa there remain unresolved issues. than focusing on individual pathovars. Although the name The bacterial pathogen X. campestris pv. musacearum (Yirgou [X. vasicola pv. vasculorum] (Vauterin et al. 1995) is invalid, this and Bradbury 1968) Dye 1978 presents a major threat to cultivation name has come to be understood by the community to represent a of banana and enset crops in central and eastern Africa, where it meaningful biological grouping; that is, a set of X. campestris pv. causes banana Xanthomonas wilt (BXW) and enset Xanthomonas vasculorum strains that are biochemically and phylogenetically wilt (EXW). Originally described as X. musacearum (Yirgou and similar to X. vasicola and pathogenically distinct. Therefore, we Bradbury 1968), this pathogen was first isolated in Ethiopia from propose a formal description of X. vasicola pv. vasculorum pv. nov. enset and banana in the 1960s and early 1970s, respectively (Yirgou that adheres to the Standards (Young et al. 2001), to harmonize the and Bradbury 1968, 1974). Symptoms consistent with EXW were formal nomenclature with that which is in common use. reported for Ethiopia as early as the 1930s (Castellani 1939). Adoption of competing classifications and invalid names has led However, only in the 21st century did the disease establish in the to the potentially confusing use of three different valid species banana-growing areas of Burundi, Democratic Republic of Congo, names (X. campestris, X. axonopodis, and X. vasicola) to describe Kenya, Rwanda, Tanzania, and Uganda (Biruma et al. 2007; Carter this group of bacteria in the literature. For example, various authors et al. 2010; Ndungo et al. 2006; Reeder et al. 2007; Tushemereirwe have referred to the strain NCPPB 1326 as X. campestris pv. et al. 2004). In this region around the Great Lakes of eastern and vasculorum, X. axonopodis pv. vasculorum (to which the strain central Africa, BXW disease severely challenges the livelihoods clearly does not belong), and [X. vasicola pv. vasculorum] (Lewis and food security of millions (Blomme et al. 2013, 2017; Biruma Ivey et al. 2010; Qhobela and Claflin 1992; Qhobela et al. 1990; et al. 2007; Nakato et al. 2018; Shimwela et al. 2016; Tinzaara et al. 2016). There is confusion in the literature about the taxonomy of this pathogen, with some authors adopting the invalid name [X. vasicola pv. musacearum]. The use of this name was motivated TABLE 1. Classification of strains previously assigned to Xanthomonas by studies suggesting a close relationship between X. campestris campestris pv. vasculorum pv. musacearum (Yirgou and Bradbury 1968) Dye 1978b and Vauterin Dookun Peros´ Current X. vasicola pv. holcicola (Elliott 1930) Vauterinet al. 1995 based on (Vauterin et al. (Dookun (Peros´ species fatty acid methyl ester (FAME) analysis, genomic fingerprinting Strainz 1992, 1995) et al. 2000) et al. 1994) assignation using rep-PCR and partial nucleotide sequencing of the gyrB gene NCPPB 186 Type A Group A n/a X. axonopodis (Aritua et al. 2007; Parkinson et al. 2009). In this letter, we formally NCPPB 891 Type A Group A G1 X. axonopodis propose the name X. vasicola pv. musacearum as a comb. nov., NCPPB 892 n/a Group A n/a X. axonopodis according to the Standards for naming pathovars (Young et al. 2001). NCPPB 893 n/a Group A n/a X. axonopodis NCPPB 181 Type A Group B n/a X. axonopodis The species X. vasicola Vauterin 1995 was created to encompass NCPPB 796PT Type A Group B n/a X. axonopodis X. campestris pv. holcicola (Elliott 1930) Dye 1978 and a subset of NCPPB 899 n/a Group D n/a X. axonopodis strains of X. campestris pv. vasculorum (Cobb 1894) Dye 1978 NCPPB 900 n/a Group D n/a X. axonopodis (Vauterin et al. 1995; Young et al. 1978). X. campestris pv. NCPPB 795 Type B Group C n/a X. vasicola vasculorum was split in two: the pathotype and some other strains NCPPB 889 Type B Group C n/a X. vasicola were transferred to X. axonopodis pv. vasculorum (Cobb) Vauterin, NCPPB 206 n/a Group C n/a X. vasicola NCPPB 702 n/a Group C n/a X. vasicola Hoste, Kersters & Swings, while the remainder were transferred NCPPB 795 n/a Group C n/a X. vasicola to the new species X. vasicola. NCPPB 889 n/a Group C n/a X. vasicola The background to this splitting of X. campestris pv. vasculorum NCPPB 890 n/a Group C n/a X. vasicola is that taxonomic studies revealed phenotypic and genomic NCPPB 895 n/a Group C n/a X. vasicola differences among X. campestris pv. vasculorum strains, despite NCPPB 1326 n/a Group C n/a X. vasicola their shared host ranges (Destefano´ et al. 2003; Dookun et al. 2000; NCPPB 1381 n/a Group C n/a X. vasicola Peros´ et al. 1994; Stead 1989; Vauterin et al. 1992, 1995). Vauterin z Superscript PT indicates the pathotype strain of X. campestris pv. vasculorum.

2 Wasukira et al. 2014). Type-B strains NCPPB 1326, NCPPB 702, The pathotype strain (NCPPB 2005) of X. campestris pv. and NCPPB 206 were erroneously described as X. axonopodis pv. musacearum (Yirgou and Bradbury 1968) Dye 1978b shares vasculorum (Lewis Ivey et al. 2010) though they are clearly 98.43% ANI with the type strain of X. vasicola (NCPPB 2417) members of X. vasicola. We acknowledge that our taxonomic but only 87.27% with the type strain of X. campestris (ATCC proposals will likely not eliminate mistakes such as these. 33913). Strains of X. campestris pv. vasculorum called [X. vasicola A further source of confusion is the status of strains isolated from pv. vasculorum] share >98.5% ANI with the type strain of maize for which some authors use the invalid name [X. campestris X. vasicola, as do strains of [X. campestris pv. zeae], including pv. zeae] (Coutinho and Wallis 1991; Qhobela et al. 1990). A useful SAM119 (=NCPPB 4614), which we propose as the pathotype nomenclature for this group has become more pressing since the strain of X. vasicola pv. vasculorum pv. nov. Furthermore, recent outbreak of leaf streak on corn in the United States, caused by unclassified strains NCPPB 902, NCPPB 1394, NCPPB 1395, bacteria very closely related to strains previously described as and NCPPB 1396, from Tripsacum laxum (Mulder 1961), and the [X. campestris pv. zeae]. One of these strains, NCPPB 4614 pathotype strain of X. campestris pv. arecae (Rao and Mohan 1970) (=SAM119), was suggested to be the eventual pathotype strain of Dye 1978 (NCPPB 2649) all share more than 98% ANI with the X. vasicola pv. vasculorum though no formal proposal and type strain of X. vasicola, which places them unambiguously within description was made (Korus et al. 2017; Lang et al. 2017). We X. vasicola. We note that former X. campestris pv. vasculorum concur with the previous suggestion (Lang et al. 2017) that strains strains NCPPB 900 and CFBP 5823 share much higher ANI with the classified to [X. vasicola pv. vasculorum] and [X. campestris pv. type strain of X. axonopodis (98.08 to 98.12%) than with the type zeae] (Vauterin et al. 1995) are insufficiently distinct to warrant strain of X. vasicola (90.43%), which is consistent with their separate pathovars and therefore strains previously named assignment to X. axonopodis pv. vasculorum (Cobb) Vauterin, [X. campestris pv. zeae] are assigned into the newly described Hoste, Kersters & Swings. X. vasicola pv. vasculorum pv. nov. The high ANI levels (Fig. 1) clearly delineate a species that Draft or complete sequence assemblies are now available for includes the type strain of X. vasicola (i.e., NCPPB 2417). It has been more than a thousand Xanthomonas genomes, including those of proposed that the boundary of a prokaryotic species can be delimited type strains for most species and pathotypes for most pathovars. by 95 to 96% ANI (Richter and Rossello-M´ ora´ 2009). By this crite- Genomic sequences can offer some advantages for phylogeny rion, X. campestris pv. arecae, X. campestris pv. musacearum,and and taxonomy, such as generally applicable threshold values for [X. vasicola pv. zeae] clearly fall within X. vasicola and outside X. species delineation (Glaeser and Kampfer¨ 2015; Meier-Kolthoff campestris. The next-nearest species to X. vasicola is X. oryzae;ANI et al. 2013, 2014; Richter and Rossello-M´ ora´ 2009). We calculated between the respective type strains of these two species is 91.7%. pairwise average nucleotide identity (ANI) between representative Despite the usefulness of ANI for delimiting species boundaries, Xanthomonas strains, including all available species type strains it does not include any model of molecular evolution and thus is and relevant pathotype strains and tabulated these in Figure 1. unsuited for phylogenetic reconstruction. Therefore, we used

Fig. 1. Average nucleotide identity (ANI) with type strains of Xanthomonas species. Genome sequence assemblies were obtained from GenBank and aligned against each other and ANI was calculated using the dnadiff function in MUMmer version 4 (Marc¸ais et al. 2018). Accession numbers of the genome assemblies: GCA_000774005.1, GCA_000772705.1, GCA_000277875.1, GCA_000770355.1, GCA_000277995.1, GCA_000159795.2, GCA_000278035.1, GCA_ 003111865.1, GCA_002191965.1, GCA_002191955.1, GCA_003111905.1, GCA_003111825.1, GCA_000007145.1, GCA_001660815.1, GCA_002939755.1, GCA_001401595.1, GCA_002939725.1, GCA_000724905.2, GCA_000192045.3, GCA_000488955.1, GCA_001401605.1, GCA_002018575.1, GCA_ 000482445.1, and GCA_002846205.1 (da Silva et al. 2002; Harrison and Studholme 2014; Jacques et al. 2013; Lang et al. 2017; Potnis et al. 2011; Sanko et al. 2018; Studholme et al. 2010; Vicente et al. 2017; Wasukira et al. 2014, 2012). Type strains are indicated by T. Pathotype strains that are not also type strains are indicated by PT. Strain NCPPB 4614 (=SAM119) was previously proposed as a member of [X. campestris pv. zeae] and also as the pathotype for [X. vasicola pv. vasculorum] and is indicated with an asterisk.

3 RaxML via the RealPhy pipeline (Bertels et al. 2014; Stamatakis Vauterin 1995, that includes strains previously assigned to et al. 2005) to elucidate phylogenetic relationships, using a X. campestris pathovars musacearum and arecae, some strains of maximum-likelihood method based on genome-wide sequencing X. campestris pv. vasculorum, and strains collected from corn and data. This approach has the additional advantage of being based on Tripsacum laxum grass that have not been previously assigned to sequence reads rather than on genome assemblies, where the latter species nor pathovar. Here we propose that the pathovar X. vasicola may be of variable quality and completeness (Bertels et al. 2014). pv. vasculorum pv. nov. includes strains formerly classified Figure 2 depicts the phylogeny of X. vasicola based on RealPhy as X. campestris pv. vasculorum but distinguishable from analysis of genome-wide sequence data. Pathovars X. vasicola pv. X. axonopodis pv. vasculorum (Cobb) Vauterin, Hoste, Kersters & holcicola and X. campestris pv. musacearum are monophyletic, Swings by protein SDS-PAGE, FAME analysis, and DNA comprising well supported clades within the X. vasicola species. A hybridization (Vauterin et al. 1992, 1995; Yang et al. 1993). Our third well supported clade includes the four Xanthomonas strains analyses also support the transfer of X. campestris pv. arecae (Rao originating from the grass Tripsacum laxum. A fourth clade consists and Mohan 1970) Dye 1978 to X. vasicola. Although only a single of mostly X. campestris pv. vasculorum strains isolated from genome of this pathovar has been sequenced, fortunately that sugarcane but also includes X. campestris pv. vasculorum strain genome belongs to the pathotype strain of the pathovar (Bull et al. NCPPB 206 isolated from maize and several strains from maize 2010; Rao and Mohan 1970). attributed previously named [X. campestris pv. zeae]. The se- Our results are consistent with previous evidence for similarity quenced strains of [X. campestris pv. zeae] from corn (Coutinho between X. campestris pv. musacearum and strains of X. vasicola, and Wallis 1991; Lang et al. 2017; Qhobela et al. 1990; Sanko based on FAME, genomic fingerprinting with rep-PCR, and gyrB et al. 2018) are monophyletic and fall within the clade containing sequencing (Aritua et al. 2007; Parkinson et al. 2007). The formal type-B strains of X. campestris pv. vasculorum (Fig. 2). The single species description for X. vasicola Vauterin 1995 states that this sequenced strain of X. campestris pv. arecae is the pathotype and it species can be clearly distinguished by its FAME profiles (Vauterin falls immediately adjacent to the X. vasicola clade containing et al. 1995). Pathogenicity studies demonstrated phenotypic strains from corn and X. campestris pv. vasculorum type-B strains distinctiveness of X. campestris pv. musacearum (Yirgou and (Fig. 2). Bradbury 1968) Dye 1978 on banana; X. campestris pv. musacea- Strain NCPPB 206 of X. campestris pv. vasculorum was isolated rum produces severe disease on this host, whereas X. vasicola pv. from maize, in contrast to most strains of this pathovar being holcicola NCPPB 2417 and X. campestris pv. vasculorum NCPPB isolated from sugarcane. On the basis of phylogenetic analysis of 702 (which belongs to X. vasicola) induced no symptoms (Aritua DNA sequence, this strain clearly falls within X. vasicola (Wasukira et al. 2007). The species description (Vauterinet al. 1995) also states et al. 2014) and has the fatty-acid type characteristic of X. vasicola that X. vasicola is characterized by metabolic activity on the carbon (Dookun et al. 2000). However, it is phylogenetically distinct from substrates D-psicose and L-glutamic acid, and by a lack of metabolic the strains of [X. campestris pv. zeae], as illustrated in Figures 1 and activity on a range of carbon substrates (further detailed in the 2. This indicates that natural infection of maize by X. vasicola is not emended description). We are not aware that these metabolic restricted to strains of [X. campestris pv. zeae]. activities have been tested for X. campestris pv. arecae, Overall, our molecular sequence analyses strongly point to the X. campestris pv. musacearum, and [X. campestris pv. zeae]; it is existence of a phylogenetically coherent species, X. vasicola possible that the species description may need to be amended to

Fig. 2. Maximum-likelihood phylogenetic tree based on genomic sequencing reads. The maximum likelihood tree was generated using RealPhy (Bertels et al. 2014) and RaxML (Stamatakis et al. 2005). Bootstrap values are expressed as percentages of 500 trials. Type and pathotype strains are indicated by T andPT, respectively. Whole-genome shotgun sequence reads were obtained from the Sequence Read Archive (Leinonen et al. 2011) via BioProjects PRJNA73853, PRJNA163305, PRJNA163307, PRJNA31213, PRJNA374510, PRJNA374557, PRJNA439013, PRJNA439327, PRJNA439328, PRJNA439329, and PRJNA449864 (Lang et al. 2017; Sanko et al. 2018; Wasukira et al. 2014, 2012). The cartoon images denote typical host plants from which each clade of bacteria have been typically isolated. Xanthomonas vasicola pv. holcicola strain NCPPB 989 was isolated from velvet grass (Holcus sp.), while strains NCPPB 1060, NCPPB 1241, and NCPPB 2417 were isolated from sorghum (Sorghum vulgare). Strains of X. campestris pv. musacearum were isolated from banana (Musa spp.) except for NCPPB 2005, which was isolated from enset (Ensete ventricosum). Strains of X. campestris pv. zeae were isolated from maize (Zea mays), as was X. campestris pv. vasculorum NCPPB 206. The remaining X. campestris pv. vasculorum strains were isolated from sugarcane (Saccharum spp.). The pathotype strain of X. campestris pv. arecae, namely NCPPB 2649, was isolated from betel palm (Areca catechu).

4 accommodate any deviation from this definition among the members of X. vasicola) have been isolated from sugarcane and repositioned pathovars. maize and shown to infect these hosts on artificial inoculation Overall, it seems that the species X. vasicola (including (Karamura et al. 2015; Vauterin et al. 1995). X. vasicola pv. holcicola, X. campestris pv. vasculorum type-B In conclusion, analysis of available genome sequence data, strains, [X. campestris pv. zeae] strains, X. campestris pv. arecae, combined with published pathogenicity and biochemical data, and some strains isolated from T. laxum) is almost exclusively strongly supports the transfer of the X. campestris pathovars associated with monocot plants of the families Palmae and musacearum and arecae to the species X. vasicola as, respectively, Gramineae. In this respect, it is similar to its closest sibling species (i) X. vasicola pv. musacearum comb. nov. with NCPPB 2005 as the X. oryzae, whose host range is limited to Gramineae (Bradbury pathotype strain (being the type strain of X. musacearum Yirgou & 1986). The exception is a report of leaf blight and dieback in Bradbury and pathotype strain of X. campestris pv. musacearum) Eucalyptus caused by X. vasicola (Coutinho et al. 2015), remark- and (ii) X. vasicola pv. arecae comb. nov. with NCPPB 2649 as the able given the phylogenetic distance between this dicot plant and pathotype strain (being the type strain of X. arecae Rao & Mohan the usual monocot hosts of X. vasicola; the infected South African and pathotype strain of X. campestris pv. arecae). Strains NCPPB plantation was in an area where sugarcane is grown. 206, NCPPB 702, NCPPB 795, NCPPB 890, NCPPB 895, NCPPB Vauterin et al. (1995) designated the pathotype strain of 1326, NCPPB 1381, and NCPPB 4614 form a phylogenetically and X. vasicola pv. holcicola (LMG 736, NCPPB 2417, ICMP 3103, phenotypically coherent group with a distinctive host range causing and CFBP 2543) as the type strain of X. vasicola, although they did symptoms on maize and sugarcane but not on banana (Aritua et al. not use the pathovar epithet for the specific epithet of the species as 2007; Karamura et al. 2015) that falls within X. vasicola pv. is most appropriate to indicate this relationship. The natural host vasculorum pv. nov. The strains isolated from T. laxum are also range of X. vasicola pv. holcicola includes the cereal crops millet clearly within the phylogenetic bounds of X. vasicola and form a and sorghum on which it causes bacterial leaf streak (Table 2) as distinct clade but cannot be assigned to any pathovar. The previous well as wild grass belonging to the genus Holcus. The host range of proposal of [X. vasicola pv. vasculorum] was invalid due in part to the strains that Vauterin et al. (1995) called [X. vasicola pv. the lack of a designated pathotype strain (Vauterin et al. 1995). We vasculorum] is less well defined because in most of the relevant pre- designate NCPPB 4614 as the pathotype strain for this pathovar, 1995 literature it is impossible to distinguish between type-A and following the previous suggestion by Lang et al. (2017). This strain type-B of X. campestris pv. vasculorum and therefore between was previously proposed as the pathotype of [X. vasicola pv. X. axonopodis pv. vasculorum and strains belonging to X. vasicola. vasculorum] (Lang et al. 2017) and causes disease symptoms on However, X. campestris pv. vasculorum type-B strains (that is, maize and sugarcane (Lang et al. 2017) but not on banana

TABLE 2. Host ranges of the taxa discussed in this letter Additional strains in NCPPB known to be Pathotype or part of the newly Current taxon Proposed taxon type strains proposed taxon Natural hosts Hosts by inoculation X. campestris pv. X. vasicola pv. NCPPB 2649 = ICMP None Areca catechu Cocos nucifera, arecae (Rao and Mohan arecae pv. nov. 5719 = LMG 533 (Bradbury 1986; Saccharum sp. 1970) Dye 1978 Kumar 1993, 1983) (Bradbury 1986) X. campestris pv. X. vasicola pv. NCPPB 2005 = ATCC NCPPB 2251; NCPPB Ensete ventricosum, Saccharum sp. musacearum (Yirgou musacearum pv. nov. 49084 = CFBP 4378; NCPPB 4379; Musa sp. (Bradbury (Karamura et al. and Bradbury 1968) 7123 = ICMP NCPPB 4380; NCPPB 1986), Tripsacum 2015), Zea mays Dye 1978 2870 = LMG 785 4381; NCPPB 4383; sp. (E. Wicker, (Karamura et al. NCPPB 4384; NCPPB unpublished 2015; Aritua 4386; NCPPB 4387; observation) et al. 2007) NCPPB 4388; NCPPB 4389; NCPPB 4390; NCPPB 4391; NCPPB 4392; NCPPB 4393; NCPPB 4394; NCPPB 4395; NCPPB 4433; NCPPB 4434 [X. vasicola pv. zeae X. vasicola pv. NCPPB 4614 = SAM119 None Zea mays (Coutinho Sorghum sp. Coutinho and vasculorum pv. nov. and Wallis 1991) (Lang et al. 2017) Wallis 1991] [X. vasicola pv. zeae Qhobela et al. 1990] X. vasicola pv. holcicola X. vasicola pv. NCPPB 2417 = CFBP NCPPB 989; NCPPB Panicum miliaceum, Echinochloa frumentacea, (Elliott 1930) Vauterin holcicola (Elliott 2543 = ICMP 1060; NCPPB 1241; Sorghum spp., Pennisetum typhoides, et al. 1995 (synonym 1930) Vauterin et al. 3103 = LMG 736 NCPPB 2417; NCPPB Zea mays Setaria italica of X. campestris pv. 1995 2930; NCPPB 3162 (Bradbury 1986) (Bradbury 1986) holcicola) X. campestris pv. X. vasicola pv. NCPPB 4614 = SAM119 NCPPB 206; NCPPB Saccharum spp., Saccharum spp., vasculorum type vasculorum pv. nov. 702; NCPPB 795; Zea mays, Zea mays B = [X. vasicola NCPPB 889; NCPPB Eucalyptus grandis (Karamura et al. 2015) pv. vasculorum 890; NCPPB 895; (Coutinho et al. (Vauterin et al. 1995)] NCPPB 1326; NCPPB 2015; Bradbury 1381; NCPPB 4614 1986; Vauterin et al. 1995) Xanthomonas sp. X. vasicola Vauterin Not applicable NCPPB 1394; Tripsacum laxum Not known et al. 1995 NCPPB 1395; NCPPB (Mulder 1961), 1396; NCPPB 902 Vetiveria zizanoides (Kumar 1983, 1993)

5 (Supplementary Fig. S1). Furthermore, given that strains from corn range includes Areca catechu (areca nut). Bradbury (1986) reports formerly described by the invalid name [X. campestris pv. zeae] are the artificial host range to include Cocos nucifera (coconut). Needle members of X. vasicola and have host ranges that cannot be prick into sugar cane produced limited streaks, but the bacteria did distinguished from the pathotype strain of X. vasicola pv. multiply to some extent and could be reisolated. Disease: leaf stripe. vasculorum pv. nov., we propose that these strains are members Long, narrow water-soaked lesions, becoming dark brown or black of this pathovar. Phylogenetic data support this as the corn strains with age. It is not known if the strains being transferred to this taxon represent a subclade within strains of X. campestris pv. vasculorum conform to the species description for metabolic activity. that fall within the emended X. vasicola. Pathotype strain: LMG 533 (= ICMP 5719 = NCPPB 2649 = PDDCC 5791). X. vasicola pv. musacearum (Yirgou & Bradbury) Dye EMENDED DESCRIPTION OF X. VASICOLA 1978 comb. nov. =X. campestris pv. musacearum (Yirgou & VAUTERIN ET AL. 1995 Bradbury) Dye 1978. Description as for the species and this pathovar is identified to species and distinguished on the basis of The characteristics are as described for the genus and the phytopathogenic specialization and is distinct from other pathovars species (Vauterin et al. 1995) extended with phylogenetic data from by its gyrB gene sequence (Parkinson et al. 2009) and genome-wide this study. The species can be clearly distinguished from other sequence analysis. Gelatin slowly liquefied, starch not hydrolyzed. xanthomonads by multilocus sequence analysis (MLSA) and whole Growth quite rapid and very mucoid when cultured on yeast- genome sequence analysis with members having more than 98% peptone-sucrose agar based media for 48 h at 28°C. According to ANI with the type strain. SDS-PAGE protein and FAME profiles Bradbury (1986), the natural hosts include Ensete ventricosum have been shown to be distinguishing for some pathovars (Aritua (enset) and Musa spp. (banana). Additional hosts by inoculation: et al. 2007; Vauterin et al. 1992; Yang et al. 1993) by the presence of Saccharum sp. (sugarcane) and Zea mays (maize) and disease is metabolic activity on the carbon substrates D-psicose and L- exhibited as a bacterial wilt where leaves wilt and wither; yellowish glutamic acid, and by a lack of metabolic activity on the carbon bacterial masses are found in vascular tissue and parenchyma. 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